Protein capable of binding specifically to immunoglobulin, and immunoglobulin-binding affinity ligand

ABSTRACT

An object of the present invention is to create a novel engineered Protein A ligand having better antibody dissociation properties in the acidic condition compared with known engineered Protein A ligands. The present invention provides a protein having an affinity for an immunoglobulin, including an amino acid sequence obtained by introducing, into an amino acid sequence derived from any of E, D, A, B and C domains of Protein A, at least one amino acid substitution at any one or more of amino acid residues corresponding to positions 31 to 37 of the A, B and C domains (positions 29 to 35 of the E domain, positions 34 to 40 of the D domain), which are conserved in all the domains, the protein having a lower affinity for an Fab region of an immunoglobulin than a protein having the amino acid sequence before introduction of the substitution.

TECHNICAL FIELD

The present invention relates to a protein capable of specificallybinding to an antibody, an affinity separation matrix containing theprotein as an immunoglobulin-binding affinity ligand, and a method forseparating and purifying or adsorbing and removing an antibody by theuse of the matrix.

BACKGROUND ART

Antibodies specifically bind to substances called antigens, and detoxifyand remove antigen-containing factors with the cooperation of otherbiomolecules and cells. The name “antibody” is particularly based onsuch an antigen-binding ability, and is also referred to as“immunoglobulin (Ig)” as a chemical name.

Recent developments in genetic engineering, protein engineering, andcell technology have accelerated the development of antibody drugs,which are pharmaceuticals utilizing the abilities of antibodies. Sincethe antibody drugs more specifically attack a target molecule thanconventional pharmaceuticals, use thereof is expected to further reduceside effects and to produce higher therapeutic effects. In fact, thesedrugs contribute to improvement in various disease conditions.

The quality of antibody drugs is thought to largely depend on the puritycompared with the quality of other recombinant protein pharmaceuticalsbecause the doses of these antibody drugs to the body are very large. Inorder to produce a high purity antibody, techniques using an adsorbingmaterial that contains a ligand molecule capable of specifically bindingto an antibody (e.g. affinity chromatography) are commonly employed.

Antibody drugs developed so far are generally monoclonal antibodies.These antibodies are mass produced by recombinant cell-culturetechnology or the like. The “monoclonal antibodies” refer to antibodiesproduced by clones of a single antibody-producing cell. Almost allantibody drugs currently available on the market are classified intoimmunoglobulin G (IgG) subclasses based on their molecular structures.One well-known example of immunoglobulin-binding proteins havingaffinities for IgG antibodies is Protein A. Protein A is a cell wallprotein produced by the gram-positive bacterium Staphylococcus aureusand contains a signal sequence S, five immunoglobulin-binding domains (Edomain, D domain, A domain, B domain and C domain) and a cellwall-anchoring domain known as XM region (Non Patent Literature 1). Inthe initial purification step (capturing step) in the process ofantibody drug manufacture, affinity chromatography columns where ProteinA is immobilized as a ligand on a water-insoluble carrier (hereinafter,referred to as Protein A columns) are commonly used (Non PatentLiteratures 1 to 3).

Various techniques for improving the performance of Protein A columnshave been developed. Various technological developments in ligands havealso been made. Initially, wild-type Protein A has been used as aligand, but currently, recombinant Protein A altered by proteinengineering is used as a ligand in many techniques for improving thecolumn performance.

Typical examples of such recombinant Protein A include recombinantProtein A without the XM region that does not bind to immunoglobulins(rProtein A Sepharose (registered trademark) available from GE healthcare, Japan). Currently, columns containing as a ligand recombinantProtein A without the XM region are widely used for industrial purposesbecause these columns have an advantage of suppressing non-specificadsorption of proteins compared with conventional ones.

Also known are inventive techniques in which a recombinant Protein Aobtained by introducing a single Cys mutation into Protein A (PatentLiterature 1) or a recombinant Protein A obtained by introducing aplurality of Lys mutations (Patent Literature 2) is used as a ligand.These techniques are effective in immobilization of ligands on awater-insoluble carrier and are advantageous in terms of theantibody-binding capacity of columns and for reducing leakage of theimmobilized ligands.

Another well-known technique is using, as an engineered recombinantProtein A ligand, an engineered domain obtained by introducing mutationinto the B domain (this engineered domain is referred to as Z domain)(Non Patent Literatures 1 and 4 and Patent Literature 3). Specifically,the Z domain is an engineered domain containing a substitution of Alafor Gly at position 29 of the B domain. In the Z domain, a substitutionof Val for Ala at position 1 of the B domain is also contained, and thismutation is intended to facilitate genetic engineering preparation of agene encoding multiple connected domains and does not affect the domainfunctions (for example, a variant containing a substitution of Ala forVal at position 1 of the Z domain is used in an example of PatentLiterature 4).

The Z domain is known to be more alkali resistant than the B domain andhas an advantage in reuse of a column through washing with an alkalinesolution having high bactericidal and cleansing effect. PatentLiteratures 5 and 6 disclose inventive ligands derived from the Zdomain, containing a substitution of another amino acid for Asn in orderto impart higher alkali resistance, and these ligands are already usedfor industrial purposes.

As described above, it is widely known that the substitution of Ala forGly at position 29 of a immunoglobulin-binding domain (E, D, A, B or Cdomain) of Protein A is a useful mutation strategy. In fact, the priorProtein A engineering technologies developed after the disclosure of the“G29A” mutation in 1987 involve the “G29A” mutation (Patent Literatures2, 4 and 6).

Another feature of the Z domain is its reduced binding ability to theFab region of immunoglobulins (Non Patent Literature 5). This featureadvantageously facilitates dissociation of an antibody binding to thedomain with the use of an acid (Non Patent Literature 1 and PatentLiterature 7). If an antibody readily dissociates, an eluate having ahigher concentration of antibodies can be recovered using less eluant.Recent developments in antibody drug manufacture have increased the cellculture production capacity beyond 10,000 liters per batch, and in thepast few years the antibody expression level has been improved up tonearly 10 g/L (Non Patent Literature 6). These developments havenaturally created a need for scale-up of the processing capacity of thedownstream purification process, and there is a very large demand fortechnical improvement in order to recover an eluate having a higherconcentration of antibodies by using less eluant.

In addition to the Z domain, engineered Protein A ligands derived fromthe C domain of Protein A have also been studied (Patent Literature 4).These ligands characteristically take advantage of the inherent highalkali resistance of the wild-type C domain and have been receivingattentions as new alternative base domains to the Z domain preparedbased on the B domain. However, our studies on the C domain haverevealed a disadvantage that it is difficult to dissociate an antibodybinding to the C domain with the use of an acid. The C domain, as taughtin Non Patent Literature 2 and Patent Literature 4, has a strong bindingability to the Fab region of immunoglobulins, and this ability ispresumed to make it difficult to dissociate the antibody with an acid.In order to overcome this disadvantage, we have examined a C domainvariant containing a substitution of Ala for Gly at position 29 for itsantibody dissociation properties in the acidic condition. The resultshave revealed that the antibody tends to more readily dissociate fromthe domain variant than the wild-type C domain, but its properties arenot enough yet.

At present, the “G29A” mutation is the only one mutation which is knownto cause an antibody to readily dissociate from theimmunoglobulin-binding domains of Protein A, as described above. The“G29A” mutation has the above-mentioned advantages as well as readydissociation of an antibody, and further technical improvement bymutations at positions other than position 29 is demanded. However,there are so far no reports of mutations at positions other thanposition 29 which enable readier dissociation of an antibody.

CITATION LIST Patent Literature

-   Patent Literature 1: U.S. Pat. No. 6,399,750-   Patent Literature 2: JP 2007-252368 A-   Patent Literature 3: U.S. Pat. No. 5,143,844-   Patent Literature 4: JP 2006-304633 A-   Patent Literature 5: European Patent No. 1123389-   Patent Literature 6: WO 03/080655-   Patent Literature 7: U.S. Publication No. 2006/0194950

Non Patent Literature

-   Non Patent Literature 1: Hober S. et al., “J. Chromatogr. B”, 2007,    Vol. 848, pp. 40-47-   Non Patent Literature 2: Low D. et al., “J. Chromatogr. B”, 2007,    Vol. 848, pp. 48-63-   Non Patent Literature 3: Roque A. C. A. et al., “J. Chromatogr. A”,    2007, Vol. 1160, pp. 44-55-   Non Patent Literature 4: Nilsson B. et al., “Protein Engineering”,    1987, Vol. 1, pp. 107-113-   Non Patent Literature 5: Jansson B. et al., “FEMS Immunology and    Medical Microbiology”, 1998, Vol. 20, pp. 69-78-   Non Patent Literature 6: Jun-ichi Inagawa et al., “Separation    process engineering”, 2008, Vol. 38, pp. 201-207

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to create a novel engineeredProtein A ligand having better antibody dissociation properties in theacidic condition compared with known engineered Protein A ligands, byintroducing mutation(s) at amino acid residue(s) at position(s) otherthan position 29.

Solution to Problem

The present inventors constructed a large number of recombinant ProteinA variant molecules containing amino acid substitutions at positionsother than position 29, recovered these variants from transformant cellsby protein engineering and genetic engineering techniques, and examinedand compared the activities of these variants. Consequently, the presentinventors completed the present invention.

Specifically, the present invention relates to a protein having anaffinity for an immunoglobulin, including an amino acid sequenceobtained by introducing, into an amino acid sequence derived from atleast one domain selected from E, D, A, B and C domains of Protein A ofSEQ ID Nos:1 to 5, at least one amino acid substitution at any one ormore of amino acid residues at positions 31 to 37 of the A, B and Cdomains, amino acid residues at positions 29 to 35 of the E domain, andamino acid residues at positions 34 to 40 of the D domain, the proteinhaving a lower affinity for an Fab region of an immunoglobulin than aprotein before introduction of the substitution.

Preferably, the amino acid sequence derived from at least one domainbefore introduction of the substitution is any of amino acid sequencesof the E, D, A, B and C domains of Protein A of SEQ ID Nos:1 to 5 andamino acid sequences of SEQ ID Nos:6 to 10 derived from the E, D, A, Band C domains of Protein A.

Preferably, the amino acid sequence derived from at least one domainbefore introduction of the substitution is an amino acid sequence of theC domain of Protein A of SEQ ID No:5 or an amino acid sequence of SEQ IDNo: 10 derived from the C domain of Protein A.

Preferably, the domain of the amino acid sequence before introduction ofthe substitution contains Ser as an amino acid residue corresponding toposition 33 of the C domain, Lys as an amino acid residue correspondingto position 35 of the C domain, Asp as an amino acid residuecorresponding to position 36 of the C domain, or Asp as an amino acidresidue corresponding to position 37 of the C domain.

Preferably, the amino acid substitution to be introduced is asubstitution of Glu, Leu or Thr for Ser corresponding to position 33 ofthe C domain, a substitution of Arg for Lys corresponding to position 35of the C domain, a substitution of Arg or Ile for Asp corresponding toposition 36 of the C domain, or a substitution of Glu for Aspcorresponding to position 37 of the C domain.

Preferably, the amino acid substitution to be introduced is asubstitution of Glu for Ser corresponding to position 33 of the Cdomain.

The present invention also relates to a multi-domain protein, includingtwo or more proteins described above, connected together.

The proteins connected together are preferably different from oneanother, and the number of the proteins connected together is preferably2 to 5.

The present invention further relates to a DNA encoding any one of theseproteins.

Preferably, in the DNA, ligated base sequences each including the domainhave a sequence identity of not higher than 90% to one another.

The present invention further relates to a vector containing the DNA.

The present invention further relates to a transformant which isobtainable by transformation of a host with the vector.

Preferably, the host for the transformant is a gram-positive bacterium.

The gram-positive bacterium is preferably a bacterium of Brevibacillus,and the bacterium of Brevibacillus is more preferably Brevibacilluschoshinensis.

Further, the present invention relates to a method for producing any oneof the proteins, the method including utilizing the transformant or acell-free protein synthesis system using the DNA.

Preferably, the production method includes: accumulating the proteinintracellularly and/or in a periplasmic space of the transformant;and/or secreting the protein extracellularly from the transformant.

Further, the present invention relates to an affinity separation matrixcontaining: any one of the proteins as an affinity ligand, and a carriermade of a water-insoluble base material on which the protein isimmobilized.

The water-insoluble base material preferably includes a syntheticpolymer or a polysaccharide, and the polysaccharide is preferablycellulose.

The affinity separation matrix preferably binds to a protein containingan Fc region of an immunoglobulin, and it more preferably binds to animmunoglobulin G or an immunoglobulin G derivative.

Further, the present invention relates to use of the affinity separationmatrix for separation of a protein containing an Fc region of animmunoglobulin. Preferably, the separation of a protein containing an Fcregion of an immunoglobulin is aimed at separating and recovering aprotein consisting only of the Fab region of an immunoglobulin.

Advantageous Effects of Invention

The protein of the present invention has excellent antibody dissociationproperties in the acidic condition. Therefore, the use of an affinityseparation matrix in which the protein is immobilized on a carrierenables to improve separation and purification of an antibody. Whicheverdomain is used among the E, D, A, B and C domains, the protein and theaffinity separation matrix of the present invention each produce thiseffect similarly since the protein of the present invention containsmutation(s) at amino acid site(s) conserved in all the domains.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a table for comparison of the sequences of E, D, A, B and Cdomains of Protein A of Staphylococcus sp.;

FIG. 2 are sensorgrams of various GST fused C domain variants binding toa monoclonal IgG-Fab (VH3) by Biacore measurements in Example 10 of thepresent invention and Comparative Example 3; FIG. 3 is a graph of 5%dBCs of the affinity separation matrix (4) of Example 12 of the presentinvention for an antibody;

FIG. 4 show elution peak profiles obtained by antibody purificationchromatography using the affinity separation matrix (1) in Example 13 ofthe present invention and Comparative Example 1;

FIG. 5 show elution peak profiles obtained by antibody purificationchromatography using the affinity separation matrix (2) in Example 13 ofthe present invention and Comparative Example 2;

FIG. 6 show elution peak profiles obtained by antibody purificationchromatography using the affinity separation matrix (3) in Example 13 ofthe present invention and Comparative Example 1; and

FIG. 7 shows an elution peak profile obtained by antibody purificationchromatography using the affinity separation matrix (4) in Example 13 ofthe present invention.

DESCRIPTION OF EMBODIMENTS

The protein of the present invention has an amino acid sequence obtainedby introducing, into an amino acid sequence derived from at least onedomain selected from E, D, A, B and C domains of Protein A of SEQ IDNos:1 to 5, at least one amino acid substitution at any one or more ofamino acid residues at positions 31 to 37 of the A, B and C domains,amino acid residues at positions 29 to 35 of the E domain, and aminoacid residues at positions 34 to 40 of the D domain, which are conservedin all the domains, and the protein also has a lower affinity for theFab region of an immunoglobulin than a corresponding protein having theamino acid sequence before introduction of the substitution, and has anaffinity for an immunoglobulin.

Protein A is a protein containing five connected immunoglobulin-bindingdomains, i.e., immunoglobulin-binding proteins. The E, D, A, B and Cdomains of Protein A are immunoglobulin-binding domains having anability to bind to a region other than complementarity determiningregions (CDRs) of immunoglobulins. All the domains are capable ofbinding to all of the Fc and Fab regions of immunoglobulins andparticularly the Fv region in the Fab region. Although the origin ofProtein A herein is not particularly limited, Protein A ofStaphylococcus origin is preferred.

The term “protein” herein is intended to include any molecules ofpolypeptide structure and therefore include fragmented polypeptidechains and polypeptide chains linked through peptide bonds as well. Theterm “domain” refers to a higher-order protein structural unit whichconsists of several tens or hundreds of amino acid residues and is ableto fulfill a certain physicochemical or biochemical function.

The amino acid sequence derived from at least one domain means an aminoacid sequence before introduction of the mutation. This sequence is notlimited only to the wild-type amino acid sequences of the E, D, A, B andC domains of Protein A, and is intended to also include amino acidsequences partially altered by substitution, insertion, deletion andchemical modification of an amino acid residue, provided that theyencode proteins having a binding ability to the Fc region. Examples ofthe amino acid sequence derived from at least one domain include aminoacid sequences of the E, D, A, B and C domains of Protein A ofStaphylococcus origin shown in SEQ ID Nos:1 to 5, and amino acidsequences of the E, D, A, B and C domains of Protein A shown in SEQ IDNos:6 to 10. Here, the proteins having the amino acid sequences of SEQID Nos:6 to 10 are proteins having amino acid sequences obtained byintroducing a substitution of Ala for Gly corresponding to position 29of the C domain into the E, D, A, B and C domains (SEQ ID Nos:1 to 5) ofProtein A. Additionally, the Z domain, which is obtained by introducingthe A1V and G29A mutations into the B domain, is also encompassed in theamino acid sequence derived from at least one domain because it has abinding ability to the Fc region. Preferred examples of the amino acidsequence before introduction of the mutation include those of domainshaving high chemical stability and variants thereof.

The protein to which amino acid substitution(s) is/are to be introducedpreferably has a sequence identity of not less than 85%, and morepreferably not less than 90% to the wild-type amino acid sequence of anyof the E, D, A, B and C domains of Protein A, and has an ability to bindto the Fc region.

The amino acid residues conserved in all the domains refer to the sameamino acid residues that occur in equivalent locations when the aminoacid sequences of the E, D, A, B and C domains are compared. As seen inthe sequence comparison table of FIG. 1, the amino acid residues atpositions 26 to 39 of the A, B and C domains, positions 24 to 37 of theE domain, and positions 29 to 42 of the D domain are conserved in allthe domains. Specific examples of the amino acid residues conserved inall the domains include amino acid residues at positions 31 to 37 of theA, B and C domains, amino acid residues at positions 29 to 35 of the Edomain, and amino acid residues at positions 34 to 40 of the D domain.Further, Ser corresponding to position 33 of the C domain, Lyscorresponding to position 35 of the C domain, Asp corresponding toposition 36 of the C domain, and Asp corresponding to position 37 of theC domain are preferred. In particular, Ser corresponding to position 33of the C domain is more preferred although the amino acid residues arenot limited to these examples. Here, the term “corresponding” means theamino acid residues arranged in the same vertical lines when the E, D,A, B and C domains of Protein A are aligned as shown in FIG. 1.

In general, highly conserved regions of amino acid sequences areimportant for protein functions in many cases, and a mutation to such aregion is likely to cause loss of a protein function. However, althoughthe amino acid sequences of the above-mentioned regions are highlyconserved, mutations to these regions do not considerably affect thebinding ability to the Fc region of an immunoglobulin while remarkablyreducing only the binding ability to the Fab region of animmunoglobulin.

Additionally, it is known that when the amino acid sequence beforeintroduction of the mutation is that of a domain having high chemicalstability or a variant thereof, introduction of a new mutation is likelyto reduce the chemical stability because the domain consists of as smallas about 60 amino acids. For example, a variant containing asubstitution of Ala for Phe corresponding to position 30 of the C domainhas significantly reduced chemical stability against alkali (Linhult M.et al., PROTEINS: Structure, Function, and Bioinformatics, 2004, Vol.55, pp. 407-416). However, the amino acid mutations in the presentinvention can produce the above-mentioned effect while maintaining theinherent chemical stability.

The amino acid substitution means a mutation of deleting an originalamino acid and adding a different amino acid at the same position. Thedifferent amino acid to be added is not particularly limited andexamples thereof include natural proteinogenic amino acids,non-proteinogenic amino acids, and non-natural amino acids.Particularly, from the viewpoint of production by genetic engineering,natural amino acids are suitably used. Examples of natural amino acidsinclude Ala, Phe, Val, Trp, Leu, Pro, Ile, Met, Gly, Asn, Ser, Gln, Thr,Tyr, Cys, Lys, His, Asp, Glu and Arg. Particularly, Glu and Arg arepreferred. The number of amino acid substitutions is not particularlylimited, provided that the protein containing the substitution(s) haslowered affinity for the Fab region of an immunoglobulin whilemaintaining its affinity for the entire immunoglobulin. In order tomaintain the three-dimensional structure of the protein beforeintroduction of the substitution, the number of substitutions ispreferably not more than 4, and more preferably not more than 2.

With respect to the notation of substitutions of amino acid residues, asubstitution is represented by an amino acid residue of the wild-type ornon-mutated type, followed by the position number of the substitution,followed by an amino acid residue introduced by the substitution. Forexample, a substitution of Ala for Gly at position 29 is represented byG29A.

Examples of amino acid substitutions at the aforementioned amino acidsites conserved in all the domains include a substitution of Glu, Leu orThr for Ser corresponding to position 33 of the C domain, a substitutionof Arg for Lys corresponding to position 35 of the C domain, asubstitution of Arg or Ile for Asp corresponding to position 36 of the Cdomain, and a substitution of Glu for Asp corresponding to position 37of the C domain. A particularly preferred one is a substitution of Glufor Ser corresponding to position 33 of the C domain although thesubstitutions are not limited to these examples.

The protein obtained by introducing the amino acid substitution(s) has asequence identity of preferably not less than 85%, and more preferablynot less than 90% to the wild-type amino acid sequence of any of the E,D, A, B and C domains of Protein A, and has a binding ability to the Fcregion.

The protein of the present invention may be a protein consisting only ofa single domain containing amino acid substitution(s), or may be amulti-domain protein including two or more of the proteins connectedtogether.

In the case of a multi-domain protein, the proteins connected togethermay be proteins derived from the same domain (i.e. homopolymer such ashomodimer or homotrimer), or proteins derived from different domains(i.e. heteropolymer such as heterodimer or heterotrimer). The number ofproteins connected together is preferably 2 or more, more preferably 2to 10, and still more preferably 2 to 5.

In the multi-domain protein, examples of the connection betweenmonomeric proteins or single domains include, but are not limited to, aconnection without any linker amino acid residues and a connection byone or more amino acid residues. The number of amino acid residues usedfor the connection is not particularly limited. The connection methodand the number of connections are also not particularly limited as longas they do not cause destabilization of the three-dimensional structureof the monomeric proteins.

Also included within the scope of the protein of the present inventionare fusion proteins in which such a protein or multi-domain protein asdescribed above, as one component, is fused with another protein havinga different function. Examples of the fusion proteins include, but arenot limited to, fusion proteins with albumin, GST (glutathioneS-transferase) or MBP (maltose-binding protein). Proteins expressed inthe form of fusion proteins with GST or MBP can be easily purified.Additionally, fusion proteins with a nucleic acid (e.g. DNA aptamer), adrug (e.g. antibiotic substance) or a polymer (e.g. PEG (polyethyleneglycol)) are also included within the scope of the protein of thepresent invention, provided that they produce the same effect of thepresent invention.

The present invention also relates to a DNA encoding the protein. TheDNA may be any DNA having a base sequence which can be translated intothe amino acid sequence of the protein of the present invention. Such abase sequence can be obtained by common known techniques, for example,using polymerase chain reaction (hereinafter, abbreviated as PCR)technology. Alternatively, the base sequence can be synthesized by knownchemical synthesis techniques or is available from DNA libraries. Acodon in the base sequence may be replaced with a degenerate codon, thatis, the base sequence is not necessarily the same as the original basesequence, provided that the translated amino acids are the same as thoseencoded by the original base sequence.

The DNA of the present invention can be obtained by site-directedmutagenesis of a known DNA encoding a wild-type domain of Protein A or avariant thereof. The site-directed mutagenesis can be carried out asfollows, using recombinant DNA technology, PCR technology or the like.

In the case of mutagenesis by recombinant DNA technology, for example,if there are suitable restriction enzyme recognition sequences on bothsides of a mutagenesis target site in the gene encoding the protein ofthe present invention, cassette mutagenesis can be used in which aregion containing the mutagenesis target site is removed by cleavingthese restriction enzyme recognition sites with the restriction enzymes,and then a DNA fragment containing mutation only at the target site,prepared by a method such as chemical synthesis, is inserted.

In the case of site-directed mutagenesis by PCR, for example, doubleprimer mutagenesis can be used in which PCR is carried out using adouble-stranded plasmid encoding the protein as a template, and twokinds of synthetic oligo primers containing mutation, complementary tothe + and − strands.

In the case of a DNA encoding the multi-domain protein, it can beproduced by ligating the desired number of DNAs each encoding themonomeric protein (single domain) of the present invention in tandem.Ligation to produce a DNA encoding the multi-domain protein can beaccomplished, for example, by introducing a suitable restriction enzymesite into DNA sequences, fragmenting the DNA sequences with therestriction enzyme, and ligating the obtained double-stranded DNAfragments using a DNA ligase. Only one restriction enzyme recognitionsite may be introduced or restriction enzyme sites of different typesmay be introduced. Alternatively, such a DNA encoding the multi-domainprotein may be produced, for example, by applying the aforementionedmutagenesis technologies to a DNA encoding Protein A (for example, WO06/004067). If the base sequences encoding monomeric proteins in the DNAencoding the multi-domain protein are the same, homologous recombinationmay occur in host cells. Therefore, the ligated DNAs each encoding themonomeric protein preferably have a base sequence identity of not higherthan 90%, and more preferably not higher than 85% to one another.

The vector of the present invention contains a base sequence encodingthe protein or the multi-domain protein, and a promoter that is operablylinked to the base sequence to function in host cells. Typically, thevector can be constructed by linking or inserting the protein-encodingDNA to a vector.

The vector to which the gene is to be inserted is not particularlylimited, provided that it is capable of autonomous replication in hostcells. As such a vector, a plasmid DNA or a phage DNA can be used. Forexample, in the case of using Escherichia coli host cells, examples ofthe vector to which the gene is to be inserted include pQE seriesvectors (QIAGEN), pET series vectors (Merck), and pGEX series vectors(GE health care, Japan). In the case of using Brevibacillus host cells,examples of the vector include the known Bacillus subtilis vectorpUB110, and pHY500 (JP H02-31682 A), pNY700 (JP H04-278091 A),pNU211R2L5 (JP H07-170984 A), and pHT210 (JP H06-133782 A), and theshuttle vector pNCMO2 between Escherichia coli and bacteria ofBrevibacillus (JP 2002-238569 A).

A transformant can be obtained by transformation of a host with thevector. The host is not particularly limited, and preferred examples ofthose suited for low-cost mass production include Escherichia coli,Bacillus subtilis and bacteria (eubacteria) of genera includingBrevibacillus, Staphylococcus, Streptococcus, Streptomyces, andCorynebacterium. More preferred are gram-positive bacteria such asBacillus subtilis and bacteria of genera including Brevibacillus,Staphylococcus, Streptococcus, Streptomyces, and Corynebacterium. Stillmore preferred are bacteria of Brevibacillus, which are known to be usedfor mass production of Protein A (WO 06/004067).

The bacteria of Brevibacillus are not particularly limited and examplesthereof include Brevibacillus agri, B. borstelensis, B. brevis, B.centrosporus, B. choshinensis, B. formosus, B. invocatus, B.laterosporus, B. limnophilus, B. parabrevis, B. reuszeri, and B.thermoruber. Preferred examples include Brevibacillus brevis 47(JCM6285), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis47-5Q (JCM8970), Brevibacillus choshinensis HPD31 (FERM BP-1087) andBrevibacillus choshinensis HPD31-OK (FERM BP-4573). Mutant strains (orderivative strains) such as protease-deficient strains, high-expressionstrains and sporulation-deficient strains of the bacteria ofBrevibacillus may be used according to purposes such as improvement inyields. Specifically, the protease mutant strain HPD31-OK ofBrevibacillus choshinensis (JP H06-296485 A) and thesporulation-deficient strain HPD31-SP3 of Brevibacillus choshinensis (WO05/045005) derived from Brevibacillus choshinensis HPD31 may be used.

Examples of the method for transfecting host cells with the vectorinclude, but are not limited to, a method using calcium ions, anelectroporation method, a spheroplast method, a lithium acetate method,an Agrobacterium infection method, a particle gun method, and apolyethylene glycol method. In order for the obtained gene to expressits function in the host cells, for example, a method includingincorporation of the gene obtained in the present invention into thegenome (chromosome) may be used.

The protein can be produced utilizing the transformant or a cell-freeprotein synthesis system using the DNA.

In the case of using the transformant for the production of the protein,the transformant may be cultured in a medium to produce and accumulatethe protein of the present invention in the cultured cells (includingthe periplasmic space thereof) or in the culture liquid(extracellularly). Thus, the desired protein can be collected from theculture.

Also in the case of using the transformant for the production of theprotein, the protein can be accumulated intracellularly and/or theperiplasmic space of the transformant. In the case where the expressedprotein is thus intracellularly accumulated, it is advantageous in thatthe protein can be protected from oxidation, and side reactions withmedium components can be avoided. In the case where the expressedprotein is accumulated in the periplasmic space, degradation by anintracellular protease can advantageously be inhibited. Alternatively,the protein may be secreted extracellularly from the transformant in theproduction of the protein. This technique advantageously providesproduction cost savings because processes such as cell disruption andextraction are not required.

The transformant of the present invention can be cultured in a medium inaccordance with a common method for culturing host cells. The medium tobe used for culturing the obtained transformant is not particularlylimited, provided that it enables high yield production of the proteinat high efficiency. Specifically, carbon and nitrogen sources such asglucose, sucrose, glycerol, polypeptone, meat extracts, yeast extracts,and casamino acids can be used. In addition, the medium may besupplemented, as required, with inorganic salts such as potassium salts,sodium salts, phosphates, magnesium salts, manganese salts, zinc salts,and iron salts. In the case of auxotrophic host cells, nutritionalsubstances necessary for their growth may be added to the medium.Moreover, antibiotics such as penicillin, erythromycin, chloramphenicol,and neomycin may be optionally added.

Furthermore, any one or more of a variety of known protease inhibitorssuch as phenylmethane sulfonyl fluoride (PMSF), benzamidine,4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), antipain,chymostatin, leupeptin, pepstatin A, phosphoramidon, aprotinin, andethylenediaminetetraacetic acid (EDTA), and other commercially availableprotease inhibitors may be added at appropriate concentrations in orderto inhibit degradation or molecular-size reduction of the desiredprotein by a host-derived protease present inside or outside the cells.

In order to assist accurate folding of the protein of the presentinvention, a molecular chaperone such as GroEL/ES, Hsp70/DnaK, Hsp90 andHsp104/ClpB may be used. For example, such a molecular chaperone can becoexpressed with the protein of the present invention or can be allowedto coexist with the protein of the present invention by combining into afusion protein or the like. Further examples of techniques for accuratefolding of the protein of the present invention include, but are notlimited to, addition of an additive for assisting accurate folding tothe medium; and culturing at low temperatures.

Examples of media for culturing transformant cells obtained from anEscherichia coli host include LB medium (1% triptone, 0.5% yeastextract, 1% NaCl) and 2×YT medium (1.6% triptone, 1.0% yeast extract,0.5% NaCl).

Examples of media for culturing transformant cells obtained from aBrevibacillus host include TM medium (1% peptone, 0.5% meat extract,0.2% yeast extract, 1% glucose, pH 7.0) and 2SL medium (4% peptone, 0.5%yeast extract, 2% glucose, pH 7.2).

The protein of the present invention is accumulated in the culturedcells (including the periplasmic space thereof) or in the culture liquid(extracellularly) by aerobically culturing the cells at a temperature of15° C. to 42° C., preferably 20° C. to 37° C., for several hours toseveral days in an aeration-stirring condition prior to recovery. Insome cases, the cells may be cultured anaerobically without aeration.

In the case where the recombinant protein is produced and secreted, theproduced recombinant protein can be recovered, after culturing thecells, by separating the cultured cells from the supernatant containingthe secreted protein by a common separation method such ascentrifugation and filtration.

Also in the case where the protein is accumulated in the cultured cells(including the periplasmic space), the protein produced and accumulatedin the cells can be recovered, for example, by collecting the cells fromthe culture liquid by centrifugation, filtration or the like, and thendisrupting the cells by sonication, a French press treatment or thelike, and/or adding an agent for making the protein soluble, such as asurfactant.

In the case where the protein of the present invention is produced by acell-free protein synthesis system, the cell-free protein synthesissystem is not particularly limited, and examples thereof includesynthesis systems derived from procaryotes, plant cells, and higheranimal cells.

Purification of the protein of the present invention can be accomplishedby any one or an appropriate combination of techniques such as affinitychromatography, cation or anion exchange chromatography and gelfiltration chromatography.

Examples of techniques to confirm whether the obtained purified productis the desired protein include common techniques such as SDSpolyacrylamide gel electrophoresis, N-terminal amino acid sequenceanalysis and Western blot analysis.

An affinity separation matrix can be prepared by immobilizing as anaffinity ligand the protein produced by the above method on a carriermade of a water-insoluble base material. The term “affinity ligand”refers to a substance (functional group) that selectively captures(binds to) a target molecule in a mixture of molecules due to a specificaffinity between molecules, typically, antigen-antibody bindingaffinity, and refers herein to a protein that specifically binds to animmunoglobulin. The term “ligand” as used alone herein is synonymouswith the “affinity ligand”.

Examples of the carrier made of a water-insoluble base material used inthe present invention include inorganic carriers such as glass beads andsilica gel; organic carriers such as synthetic polymers (e.g.cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linkedpolyacrylamide, cross-linked polystyrene) and polysaccharides (e.g.crystalline cellulose, cross-linked cellulose, cross-linked agarose,cross-linked dextran); and composite carriers of combinations of thesecarriers such as organic-organic or organic-inorganic compositecarriers.

Examples of commercial products thereof include GCL2000 (porouscellulose gel), Sephacryl S-1000 (prepared by covalently cross-linkingallyl dextran with methylenebisacrylamide), Toyopearl (methacrylatecarrier), Sepharose CL4B (cross-linked agarose carrier) and Cellufine(cross-linked cellulose carrier). It should be noted that thewater-insoluble carrier usable in the present invention is not limitedonly to the carriers listed above.

In view of the purpose and method of usage of the affinity separationmatrix, the water-insoluble carrier used in the present inventiondesirably has a large surface area and is preferably a porous matrixhaving a large number of fine pores of a suitable size. The carrier maybe in any form such as beads, monolith, fiber, or film (including hollowfiber), and any form can be selected appropriately.

Immobilization of the ligand on the carrier may be accomplished, forexample, by a conventional coupling method utilizing an amino, carboxylor thiol group of the ligand. Examples of such a coupling method includeimmobilization methods including activation of the carrier by reactingthe carrier with cyanogen bromide, epichlorohydrin, diglycidyl ether,tosyl chloride, tresyl chloride, hydrazine, sodium periodate, or thelike (or introduction of a reactive functional group into the carriersurface), followed by a coupling reaction between the carrier and acompound to be immobilized as the ligand; and immobilization methodsinvolving condensation and crosslinking by adding a condensation reagentsuch as carbodiimide or a reagent containing a plurality of functionalgroups in the molecule, such as glutaraldehyde, to a system containingthe carrier and a compound to be immobilized as the ligand.

A spacer molecule consisting of a plurality of atoms may also beintroduced between the ligand and the carrier, or alternatively, theligand may be directly immobilized on the carrier. That is, forimmobilization, the protein of the present invention may be chemicallymodified, or may incorporate an additional amino acid residue useful forimmobilization. Examples of amino acids useful for immobilizationinclude amino acids containing, in a side chain, a functional groupuseful for a chemical reaction for immobilization, and specificallyinclude Lys which contains an amino group in a side chain, and Cys whichcontains a thiol group in a side chain. In the nature of the presentinvention, the matrix containing the protein of the present invention asa ligand immobilized therein should also have the effect of the proteinof the present invention, and the matrix is also included within thescope of the present invention even if the protein is modified oraltered in any manner for immobilization.

The affinity separation matrix is thus obtained by immobilizing theprotein of the present invention. Therefore, due to the activity of theprotein of the present invention, per se, the affinity separation matrixis capable of binding to a protein containing the Fc region of animmunoglobulin. Accordingly, the protein containing the Fc region of animmunoglobulin can be separated and purified by an affinity columnchromatography purification method using the protein and the affinityseparation matrix of the present invention. The term “protein containingthe Fc region of an immunoglobulin” refers to a protein containing anypart of the Fc region to which Protein A binds. It should be noted thatthe protein is not required to contain the entire Fc region as long asProtein A can binds thereto.

Examples of the protein containing the Fc region of an immunoglobulininclude, but are not limited to, immunoglobulin Gs and immunoglobulin Gderivatives. Specific examples of the protein containing the Fc regionof an immunoglobulin include IgG of the VH3 subfamily, in particular,human IgG of the VH3 subfamily (monoclonal antibody). The protein andthe affinity separation matrix of the present invention preferably havea lower affinity for the Fab region of IgG of the VH3 subfamily than aprotein before introduction of the mutation, and at the same time theypreferably have an affinity for the Fc region of IgG subtypes 1, 2 and4. Nearly half of human VH germ line genes belong to the VH3 subfamily.In fact, pharmaceuticals containing IgG antibodies of the VH3 subfamilyare under study and some of them are already commercially available. Inaddition, it is regarded as a known fact that in the case of using aligand maintaining the binding ability to the Fab region of animmunoglobulin of the VH3 subfamily in an affinity separation matrix forantibody purification, the remaining binding ability adversely affectsthe dissociation properties of the antibody in the acidic condition,from some literatures (Ghose S. et al., Biotechnology andbioengineering, 2005, vol. 92, No. 6). Therefore, the protein and theaffinity separation matrix of the present invention preferably have areduced binding ability to the Fab region of human IgG of the VH3subfamily.

The term “immunoglobulin G derivatives” is a generic name of alteredsynthetic proteins to which Protein A binds, such as chimericimmunoglobulin Gs in which domain(s) of human IgG is/are partiallyreplaced and fused with IgG domain(s) of another species, humanizedimmunoglobulin Gs in which CDRs (Complementarity Determining Regions) ofhuman IgG are replaced and fused with antibody CDRs of another species,immunoglobulin Gs whose Fc region has a molecularly altered sugar chain,and artificial immunoglobulin Gs in which the Fv region and the Fcregion of human IgG are fused.

The regions to be bound are loosely defined as “Fab region (inparticular, Fv region)” and “Fc region”, and the protein to which theprotein and the affinity separation matrix of the present invention bindmay be one obtained by further altering (e.g. fragmenting) the Fab or Fcregion while maintaining the three-dimensional structure of the regionto which Protein A binds by protein engineering techniques based on thethree-dimensional structure of the antibody, which is already known.

Purification of the protein containing the Fc region of animmunoglobulin using an affinity column filled with the affinityseparation matrix of the present invention is accomplished in accordancewith an affinity column chromatography purification method using aProtein A column which is already on the market (Non Patent Literature3). Specifically, a buffer containing the protein containing the Fcregion of an immunoglobulin is neutralized and the resulting solution isallowed to pass through the affinity column filled with the affinityseparation matrix of the present invention so that the proteincontaining the Fc region of an immunoglobulin is adsorbed on theaffinity separation matrix. Next, an adequate amount of a pure buffer isallowed to pass through the affinity column to wash the inside of thecolumn. At this time, the desired protein containing the Fc region of animmunoglobulin remains adsorbed on the affinity separation matrix of thepresent invention in the column. Subsequently, an acidic buffer adjustedto an adequate pH (which may contain a substance for acceleratingdissociation of the protein from the matrix) is allowed to pass throughthe column to elute the desired protein containing the Fc region of animmunoglobulin. Thus, high-level purification can be achieved.

The affinity separation matrix of the present invention can be reusedthrough a washing process in which a pure buffer (in some cases, asolution containing an appropriate modifier or organic solvent) havingan adequately strong acidity or alkalinity which does not completelyimpair the functions of the ligand compound and the carrier basematerial is allowed to pass through the matrix.

An advantageous effect of the protein of the present invention and theaffinity separation matrix utilizing this protein is that they have anaffinity for an immunoglobulin but have a reduced affinity for the Fabregion of the immunoglobulin. In general, the domains of Protein A morestrongly bind to the Fc region than to the Fab (Fv) region (Non PatentLiterature 3). Thus, the “affinity for an immunoglobulin” of Protein Aand the domains essentially refers to the affinity for the Fc region,and the degree of affinity for an immunoglobulin does not largely changewhen only the strength of binding to the Fab region is changed. Theprotein of the present invention shows a reduction in the secondaryaffinity for the Fab region which the immunoglobulin-binding domains ofProtein A inherently have. Therefore, the protein of the presentinvention has an advantageous effect of eliminating an influence of thesecondary binding by interaction with an immunoglobulin. On the otherhand, the affinity for the Fc region is maintained, and therefore theaffinity for the immunoglobulin as a whole is maintained. When theaffinity of the protein of the present invention for an immunoglobulinis evaluated as an affinity for a human immunoglobulin G drug by aBiacore system described below, the affinity constant (KA) is preferably10⁶ (M⁻¹) or higher, and more preferably 10⁷ (M⁻¹) or higher.

The affinity of the protein and the affinity separation matrix of thepresent invention for a protein containing the Fc region of animmunoglobulin can be tested by for example, but not limited to, abiosensor such as a Biacore system (GE health care, Japan) based on thesurface plasmon resonance principle.

The measurement conditions may be determined such that a binding signalemitted when Protein A binds to the Fc region of an immunoglobulin canbe detected. Specifically, the affinity can be easily evaluated bymeasurement at a temperature of 20° C. to 40° C. (constant temperature)and a neutral pH of 6 to 8.

The immunoglobulin molecule as a binding partner is not particularlylimited, provided that it allows detection of binding to the Fab region.However, fragmented immunoglobulin molecules (Fab fragments, Fvfragments) obtained by separating the Fab region from the Fc region arepreferred because binding to the Fc region is also detected if animmunoglobulin molecule containing the Fc region is used. Further, morepreferred are Fab fragments of immunoglobulins of the VH3 subfamily,which are already known to allow Protein A to bond to the Fab region.

Those skilled in the art can easily determine the difference in affinityby obtaining sensorgrams of the binding reactions with the sameimmunoglobulin molecule under the same measurement conditions, andmaking a comparison between binding parameters obtained by the analysisof proteins before and after introduction of a mutation. Here, thesequences to be compared for the difference in affinity should be thesame except for the mutated site. For example, when the sequences of theB domain and a C domain variant containing the D36R mutation are usedfor evaluation of the effect of the D36R mutation, as the amino acidsequence of a protein before introduction of the mutation and the aminoacid sequence of a protein after introduction of the mutation,respectively, the comparison between them does not make sense.

Examples of binding parameters include the affinity constant (KA) andthe dissociation constant (KD) (Nagata et al., “Real-time analysis ofbiomolecular interactions”, Springer-Verlag Tokyo, 1998, p. 41). Theaffinity constants of domain variants of the present invention for Fabcan be determined with a Biacore system by adding each domain variant toa flow channel in an experimental system that includes a sensor chipwith an Fab fragment of an immunoglobulin of the VH3 subfamilyimmobilized thereon, at a temperature of 25° C. and a pH of 7.4. Amongproteins having mutated sequences according to the present invention,those having an affinity constant (KA) reduced to not higher than ½ ofthe affinity constant of a protein having a sequence before introductionof the mutation are suitably used. The affinity constant (KA) is morepreferably reduced to not higher than ⅕, and still more preferably nothigher than 1/10. Here, it should be noted that although the affinityconstant is also described as the association constant in someliteratures, these two terms basically mean the same.

In general, C domain variants containing a substitution of Ala for Glyat position 29 have a KA for Fab of 1×10⁴ to 1×10⁵ (M⁻¹). C domainvariants containing mutation(s) according to the present invention inaddition to the substitution of Ala for Gly at position 29 and having aKA reduced to lower than 1×10⁴ (M⁻¹) are suitably used in the presentinvention. More suitably used are variants having a KA of 0.5×10⁴ (M⁻¹)or lower. The Fab used in the KA measurement may be obtained byfragmenting an immunoglobulin G into an Fab fragment and an Fc fragmentby papain; or may be prepared using a genetically engineered productionsystem that expresses only the Fab region of an immunoglobulin G.

Because of its reduced binding ability to the Fab region of animmunoglobulin, the affinity separation matrix of the present inventionexcellently dissociates an antibody in the process of eluting theantibody with an acidic solution. Specifically, since the affinityseparation matrix of the present invention allows elution under acidicelution conditions closer to neutral, damage to an antibody caused underacidic conditions can advantageously be suppressed. The acidic elutionconditions closer to neutral specifically mean conditions with a pH ofabout 3.0 to 5.0 compared with the pH range of common acidic elutionconditions of about 2.0 to 3.5. Elution under the conditions reducesdamage to an antibody (Ghose S. et al., Biotechnology andbioengineering, 2005, vol. 92, No. 6). The excellent antibodydissociation properties in the acidic condition mean, for example,dissociation under acidic elution conditions closer to neutral, or asharper elution peak profile obtained when an antibody is eluted underacidic conditions. A sharper elution peak profile in chromatographyindicates that an eluate having a higher concentration of antibodies canbe recovered using less eluant.

Additionally, the affinity separation matrix of the present inventionenables the Fab region to be separated and recovered readily as a flowthrough fraction from a mixture of a molecule containing the Fc regionand a molecule containing only the Fab region.

EXAMPLES

The following description is offered to illustrate in more detail thepresent invention based on examples, but the scope of the presentinvention is not limited to these examples.

Proteins obtained in examples are each represented by “an alphabetindicating a domain—an introduced mutation (wild for the wild-type)”.For example, the wild-type C domain of Protein A is represented by“C-wild”, and a C domain variant containing the G29A mutation isrepresented by “C-G29A”. A domain variant containing two mutationstogether is represented by indicating the two mutations together with aslash. For example, a C domain variant containing the G29A and S33Emutations is represented by “C-G29A/S33E”. A protein containing aplurality of single domains connected is represented together with aperiod (.) and the number of connected domains with “d”. For example, aprotein consisting of five connected C domain variants containing theG29A and S33E mutations is represented by “C-G29A/S33E.5d”.

[Example 1] Preparation of DNA Encoding C-G29A.5d

A base sequence encoding a protein consisting of five connected C-G29Vswas constructed by reverse translation from the amino acid sequence(C-G29V.5d, SEQ IDNo:11) of the protein. Codons were assigned such thatthe codon usage frequency of the protein was closer to the codon usagefrequency of the cell surface protein HWP, which is expressed in a largeamount in Brevibacillus choshinensis HPD31 (Ebisu S., “J. Bacteriol.”,1990, No. 172, pp. 1312-1320), and that the sequence identity betweenthe base sequences of the five domains was low. The restriction enzymerecognition sites for PstI and XbaI were also prepared on the 5′ sideand 3′ side, respectively, of the sequence encoding the five connecteddomains. The prepared DNA fragment was commissioned from Takara Bio Inc.The sequence of the prepared DNA fragment is shown as SEQ ID No:12.

The prepared DNA fragment encoding C-G29V.5d was digested with PstI andXbaI (both available from Takara Bio Inc.), and then separated andpurified by agarose gel electrophoresis. Separately, the plasmid vectorpNK3262 for Brevibacillus was digested with PstI and XbaI, and thenpurified and recovered. The recovered vector was treated with alkalinephosphatase (Takara Bio Inc.) for dephosphorylation. Both were mixed andligated with Ligation High (TOYOBO CO., LTD.). In this manner, a plasmidvector pNK3262-C-G29V.5d capable of expressing C-G29V.5d wasconstructed. Brevibacillus choshinensis FY-1 was transformed using theplasmid vector obtained by the above procedures. The transformation wasaccomplished by a known electroporation method (“Biosci. Biotech.Biochem.”, 1997, No. 61, pp. 202-203). The BrevibacilluschoshinensisFY-1 is a Phe- and Tyr-requiring strain obtained by mutatingBrevibacillus choshinensis HPD31-OK (JP H06-296485 A).

The gene encoding C-G29V.5d in the plasmid pNK3262-C-G29V.5d thusprepared was cleaved into five DNA fragments so that each fragmentcontained a codon for Val-29 of the individual domain. The domains werenumbered 1 to 5 starting from the N-terminal side. The DNA fragment forthe domain 1 was digested with PstI and NarI; the DNA fragment for thedomain 2 was digested with NarI and HindIII; the DNA fragment for thedomain 3 was digested with HindIII and MluI; the DNA fragment for thedomain 4 was digested with MluI and BglII; and the DNA fragment for thedomain 5 was digested with BglII and XbaI (NarI is available from TOYOBOCO., LTD. and the others are available from Takara Bio Inc.), eachfollowed by separation and purification using an agarose gel to give therespective DNA fragments.

Two cloning vectors pSL301 (Invitrogen) and pUC19 (Takara Bio Inc.) weredigested with the same pairs of restriction enzymes as those used forthe DNA fragments encoding the domains. The resulting fragments wereeach mixed with the corresponding DNA fragment, and they were ligatedwith Ligation High. In this manner, plasmids each containing one of thefive-divided DNA fragments were constructed. The plasmids arerepresented in correspondence to the domain numbers as pUC19-V29-d1,pUC19-V29-d2, pSL301-V29-d3, pSL301-V29-d4, and pSL301-V29-d5. Thesequences (including the restriction enzyme recognition sites) of theDNA fragments of the C-G29V.5d-encoding region are shown as SEQ IDNos:13 to 17.

Quick change mutagenesis was performed using the oligonucleotide primersof SEQ ID Nos:18 to 27, and the plasmids pUC19-V29-d1, pUC19-V29-d2,pSL301-V29-d3, pSL301-V29-d4, and pSL301-V29-d5 as templates. As aresult, plasmids pUC19-A29-d1, pUC19-A29-d2, pSL301-A29-d3,pSL301-A29-d4, and pSL301-A29-d5 were obtained, each of which containeda DNA fragment encoding C-G29A containing a substitution of Ala forVal-29 of the domain. The five fragments were sequentially ligated toone another with Ligation High. In this manner, an expression plasmidpNK3262-C-G29A.5d containing the DNA fragment (SEQ ID No:29) encodingC-G29A.5d (SEQ ID No:28) was prepared. Then, this plasmid was used fortransformation of FY-1 recombinant cells. The quick change mutagenesiswas performed in accordance with the protocol of Stratagene using PfuTurbo DNA polymerase and the methylated DNA (template DNA) cleavageenzyme DpnI (both available from Stratagene). For example, the quickchange mutagenesis was performed on the plasmid pUC19-V29-d1 containingthe DNA fragment of SEQ ID No: 13 with two synthetic DNA primers of SEQID Nos:18 and 19, thereby providing a plasmid pUC19-A29-d1 containing aDNA fragment for the domain 1 containing a substitution of Ala forVal-29.

[Example 2] Preparation of DNAs Encoding C-G29A/S33E.5d, C-G29A/D36R.5dand C-G29A/K35R/D37E.5d

The same techniques based on quick change mutagenesis as in Example 1were applied using the oligonucleotide primers of SEQ ID Nos:30 to 59,and the five plasmids each containing a C-G29A-encoding DNA fragment,pUC19-A29-d1, pUC19-A29-d2, pSL301-A29-d3, pSL301-A29-d4, andpSL301-A29-d5 prepared in Example 1, as templates. As a result, plasmidscontaining DNA fragments encoding C-G29A/S33E, C-G29A/D36R, andC-G29A/K35R/D37E were prepared.

Subsequently, the DNA fragments were ligated in the manner described inExample 1 so that an expression plasmid pNK3262-C-G29A/S33E.5dcontaining the DNA fragment (SEQ ID No:61) encoding C-G29A/S33E.5d (SEQID No:60), an expression plasmid pNK3262-C-G29A/D36R.5d containing theDNA fragment (SEQ ID No:63) encoding C-G29A/D36R.5d (SEQ ID No:62), andan expression plasmid pNK3262-C-G29A/K35R/D37E.5d containing the DNAfragment (SEQ ID No:65) encoding C-G29A/K35R/D37E.5d (SEQ ID No:64) wereprepared. These plasmids were used for transformation of FY-1recombinant cells. For ligation of the DNA fragments encoding C-G29A,the restriction enzyme HindIII was used. Since the HindIII recognitionsequence was close to the mutation site, a plasmid containing the DNAfragment encoding the unmutated domain 2 and the DNA fragment encodingthe mutated domain 3 connected through the HindIII site was prepared,and then the corresponding mutation was introduced in the domain2-encoding region. Additionally, the DNA fragment (SEQ ID No:69)encoding C-G29A/S33E.4d (SEQ ID No:68) was amplified by PCR usingpNK3262-C-G29A/S33E.5d as a template plasmid and the oligonucleotideprimers of SEQ ID Nos:66 and 67. The amplified DNA fragments weredigested with PstI and XbaI, and inserted into the vector pNK3262digested with the same enzymes. In this manner, an expression plasmidpNK3262-C-G29A/S33E.4d was prepared, and used for transformation of FY-1recombinant cells.

[Example 3] DNA Sequence Determination

The DNA base sequences of the expression plasmids obtained in Examples 1and 2 were determined using a DNA sequencer 3130xl Genetic Analyzer(Applied Biosystems). Using BigDye Terminator v. 1. 1 Cycle SequencingKit (Applied Biosystems) in accordance with the attached protocol, PCRof these plasmid DNAs for sequencing was carried out, and the sequencingproducts were purified and sequenced. The sequences of theoligonucleotide primers for sequencing were omitted here.

[Example 4] Expression of Target Protein in Expressing Recombinant Cell

The Brevibacillus choshinensis FY-1 recombinant cells obtained inExamples 1 and 2 were cultured with shaking for 3 days at 30° C. in 5 mLof 3YC medium (3% polypeptone, 0.2% yeast extract, 3% glucose, 0.01%magnesium sulfate, 0.001% iron sulfate, 0.001% manganese chloride,0.0001% zinc chloride) containing 60 μg/mL neomycin.

Each culture was centrifuged to remove cells, and the obtained culturesupernatant was subjected to cation exchange chromatography using an SPFast Flow column (GE Healthcare, Japan) to purify (partially purify) thetarget protein. Specifically, sodium acetate was added to the culturesupernatant to a final concentration of 50 mM, and hydrochloric acid wasalso added to adjust the pH to 4.0. Then, the culture supernatant wasapplied to the SP Fast Flow column equilibrated with a cation exchangebuffer A (50 mM CH₃COOH—CH₃COONa, pH 4.0). After washing the column withthe cation exchange buffer A, the target protein was eluted andseparated in the process of salt gradient elution using the cationexchange buffer A and a cation exchange buffer B (50 mMCH₃COOH—CH₃COONa, 1 M NaCl, pH 4.0).

Next, the target protein was purified by anion exchange chromatographyusing a DEAE Fast Flow column (GE Healthcare, Japan). Specifically, theseparated target protein solution was dialyzed with ultrapure water, andapplied to the DEAE Fast Flow column equilibrated with an anion exchangebuffer A (50 mM Tris-HCl, pH 8.0). After washing with the anion exchangebuffer A, the target protein was eluted and separated in the process ofsalt gradient elution using the anion exchange buffer A and an anionexchange buffer B (50 mM Tris-HCl, 0.3 M NaCl, pH 8.0). The separatedtarget protein solution was re-dialyzed with ultrapure water. In thismanner, an aqueous solution containing only the target protein wasobtained as a final purified sample.

The protein purification processes by chromatography using the columnswere carried out using an AKTAprime plus system (GE Healthcare, Japan).

[Example 5] Analysis of Affinity of Obtained Proteins for HumanImmunoglobulin G (Human IgG)

The proteins obtained in Example 4 were analyzed for affinity for animmunoglobulin by a biosensor Biacore 3000 (GE health care, Japan)utilizing surface plasmon resonance. In the present example, a humanimmunoglobulin G drug (hereinafter, referred to as human IgG) separatedfrom human plasma was used. The human IgG was immobilized on a sensorchip, and each protein was added on the chip to detect an interactionbetween them. The immobilization of the human IgG on the sensor chip CM5was carried out by amine coupling using N-hydroxysuccinimide (NHS) andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), andethanolamine was used for blocking (all the sensor chips and theimmobilization reagents are available from GE health care, Japan). Thehuman IgG solution was prepared by dissolving Gammagard (Baxter) in astandard buffer (20 mM NaH₂PO₄—Na₂HPO₄, 150 mM NaCl, pH 7.4) to aconcentration of 1.0 mg/mL. The human IgG solution was diluted to 1/100in an immobilization buffer (10 mM CH₃COOH—CH₃COONa, pH 4.5) and thehuman IgG was immobilized on the sensor chip in accordance with theprotocol attached to the Biacore 3000. A reference cell to be used as anegative control was also prepared by immobilizing ethanolamine onanother flow cell on the chip after activation by EDC/NHS. The proteinsolutions were appropriately prepared at concentrations of 10 to 1000 nMusing a running buffer (20 mM NaH₂PO₄—Na₂HPO₄, 150 mM NaCl, 0.005% P-20,pH 7.4) (solutions of three different protein concentrations wereprepared for each protein), and each protein solution was added on thesensor chip at a flow rate of 20 μL/min for 30 seconds. A sensorgram ofthe binding reaction at 25° C. was sequentially plotted during theaddition (binding phase, 30 seconds) and after the addition(dissociation phase, 60 seconds). After each sensorgram determination,the sensor chip was regenerated by adding 50 mM NaOH (for 15 seconds)(this process was performed to remove the added proteins remaining onthe sensor chip and it was confirmed that the binding activity of theimmobilized human IgG was substantially completely recovered). Thebinding rate constant (k_(on)), dissociation rate constant (k_(off)),affinity constant (K_(A)=k_(on)/k_(off)), and dissociation constant(K_(D)=k_(off)/k_(on)) were calculated by performing a fitting analysison each of the obtained binding reaction sensorgrams (the bindingreaction sensorgrams obtained by subtracting the binding reactionsensorgram of the reference cell) by using the 1:1 binding model in asoftware BIA evaluation attached to the system.

As shown in Table 1, the binding parameters of the proteins to the humanIgG were at similar levels to those of C-G29A.5d (Comparative Example1). Specifically, the affinity constants (KA) of all the proteins forthe human IgG fell in the range of 5.0×10⁷ M⁻¹ to 5.0×10⁸ M⁻¹.

TABLE 1 K_(on) (×10⁵ M⁻¹s) K_(off) (10⁻⁸ s⁻¹) K_(A) (×10⁸ M⁻¹) C-G29A.5d4.1 0.88 4.7 C-G29A/S33E.5d 3.0 1.3 2.3 C-G29A/D36R.5d 4.6 1.5 3.0C-G29A/K35R/D37E.5d 0.86 1.3 0.65

[Example 6] Preparation of Fab Fragment Derived from HumanizedMonoclonal Antibody

In the present invention, the “affinity for the Fab region” was analyzedusing an Fab fragment free from the Fc region of an immunoglobulin.

The Fab fragment was prepared by fragmenting a humanized monoclonal IgGdrug as a starting material into an Fab fragment and an Fc fragment byusing papain, and separating and purifying only the Fab fragment.

Specifically, herceptin (humanized monoclonal IgG drug available fromChugai Pharmaceutical Co., Ltd.) was dissolved in a papain digestionbuffer (0.1 M AcOH—AcONa, 2 mM EDTA, 1 mM cysteine, pH 5.5). PapainAgarose from papaya latex (papain-immobilized agarose available fromSIGMA) was added to the solution, and the resulting mixture wasincubated for about 8 hours at 37° C. while being mixed with a rotator.By ion exchange chromatography using a Resource S column (GE healthcare, Japan), the Fab fragment (hereinafter, referred to as monoclonalIgG-Fab) was separated and purified from the reaction solution(containing both the Fab fragment and the Fc fragment) which had beenseparated from the papain-immobilized agarose. More specifically, thereaction solution was diluted to pH 4.5 in an ion exchange buffer A (50mM CH₃COOH—CH₃COONa, pH 4.5), and then added to the Resource S columnequilibrated with the ion exchange buffer A. After washing the columnwith the ion exchange buffer A, the monoclonal IgG-Fab was eluted andseparated in the process of salt gradient elution using the ion exchangebuffer A and an ion exchange buffer B (50 mM CH₃COOH—CH₃COONa, 1 M NaCl,pH 4.5) (the buffer B concentration was linearly increased from 0% to50% during the process of allowing the buffers in a total amountcorresponding to the volume of 10 columns to pass through the column).

The separated monoclonal IgG-Fab solution was purified by gel filtrationchromatography using a Superdex 75 10/300 GL column (the standard bufferwas used for equilibration and separation). In this manner, a monoclonalIgG-Fab solution was obtained.

The protein purification by chromatography was performed using theAKTAprime plus system in the same manner as in Example 4.

[Example 7] Analysis of Affinity of Obtained Proteins for MonoclonalIgG-Fab

The affinity of the proteins obtained in Example 4 for the IgG-Fab wasalso analyzed using the Biacore 3000 in the same manner as in Example 5.

The monoclonal IgG-Fab obtained in Example 6 was immobilized on thesensor chip CM5, and each protein obtained in Example 4 was added on thechip to detect an interaction between them. Human serum albumin (SigmaAldrich) was immobilized on a reference cell. The immobilization of themonoclonal IgG-Fab and the human serum albumin was carried out in thesame manner as in Example 5.

Protein solutions of different concentrations (4 μM, 8 μM, 16 μM, 32 μM(32 μM samples of some proteins were not prepared)) were prepared foreach of the proteins to be measured, using a running buffer (20 mMNaH₂PO₄—Na₂HPO₄, 150 mM NaCl, 0.005% P-20, pH 7.4). Each proteinsolution was added on the sensor chip at a flow rate of 20 μL/min for 30seconds, and a sensorgram of the binding reaction at 25° C. wassequentially plotted during the addition (binding phase, 30 seconds) andafter the addition (dissociation phase, 60 seconds). After eachsensorgram determination, 10 mM NaOH was added for 30 seconds forregeneration of the sensor chip. The analysis was conducted in the samemanner as in Example 6. It should be noted that R_(max), one of bindingparameters, was regarded as a constant in the fitting analysis. TheR_(max) is the signal amount obtained when added molecules are bound toall immobilized molecules, and could not largely change in theseexperiments in which the same molecules (monoclonal IgG-Fab) areimmobilized. If the binding signal is very weak, however, a fitting isincorrectly made so that the R_(max) is regarded as an extremely smallvalue. Hence, the R_(max) was regarded as a constant in the fitting. Asshown in Table 2, the binding parameters of the proteins to themonoclonal IgG-Fab were significantly lower than those of C-G29A.5d(Comparative Example 1). Specifically, the affinity constants (KA) ofall the proteins to the monoclonal IgG-Fab were less than 1/10 of thatof C-G29A.5d.

TABLE 2 k_(on) (×10⁴ M⁻¹s) k_(off) (s⁻¹) K_(A) (×10⁵ M⁻¹) C-G29A.5d 5.80.13 4.4 C-G29A/S33E.5d 0.056 0.40 0.014 C-G29A/D36R.5d 0.22 0.39 0.057C-G29A/K35R/D37E.5d 0.23 0.41 0.059

[Example 8] Preparation of Transformant Cells Capable of ExpressingSingle-Domain Variants

In order to effectively evaluate other mutations disclosed herein inaddition to the mutations evaluated in Example 7, single-domain variantswere prepared.

The amino acid sequence (SEQ ID No:10, C-G29A. d) derived from the Cdomain of Protein A was chosen as a protein sequence to be mutated. AGST fusion protein expression vector pGEX-6P-1 (GE Healthcare, Japan)containing the DNA sequence encoding C-G29A.1d of SEQ ID No:70 was usedas a template plasmid for mutation. The template plasmid was prepared inaccordance with the description of WO 2010/110288.

Expression plasmids encoding C domain variants (C-G29A/S33L.1d,C-G29A/S33T.1d, C-G29A/D36I.1d, C-G29A/D36R.1d, and C-G29A/D37E.1d) ofSEQ ID Nos:81 to 85 were obtained by quick change mutagenesis using thetwo plasmids as templates and the oligonucleotide primers of SEQ IDNos:71 to 80. For example, the C domain variant (C-G29A/S33L.1d) of SEQID No:81 was obtained by quick change mutagenesis using the expressionplasmid (pGEX-6P-1-C-G29A.1d) containing the DNA sequence of SEQ IDNo:70 as a template and the oligonucleotide primers of SEQ ID Nos:71 and72. The DNA sequences of the obtained expression plasmids weredetermined in the same manner as in Example 3. The obtained expressionplasmids were used for transformation of E. coli HB101 (Takara Bio Inc.)in the same manner as in Example 1.

[Example 9] Expression and Purification of Single-Domain Variants

The transformants obtained in Example 8 were capable of expressing therespective variants in the form of GST fusion proteins. Each of thesetransformants was cultured in LB medium containing ampicillin at 37° C.overnight. Each culture liquid was inoculated in 2×YT medium (containingampicillin) and cultured at 37° C. for about 1 hour. IPTG(isopropyl-1-thio-β-D-galactoside) was added to a final concentration of0.1 mM, followed by further culturing at 37° C. for 18 hours. After theculturing, cells were collected by centrifugation and resuspended in PBSbuffer containing EDTA (0.5 mM). The cells were sonicated andcentrifuged to separate a supernatant fraction (cell-free extract) andan insoluble fraction. The GST fusion proteins were purified (partiallypurified) from the cell-free extracts containing the GST fusion proteinsby affinity chromatography using a GSTrap FF column (GE Healthcare,Japan), which has an affinity for GST. Each cell-free extract wasapplied to the GSTrap FF column, and the column was washed with astandard buffer (20 mM NaH₂PO₄—Na₂HPO₄, 150 mM NaCl, pH 7.4). Then, thetarget GST fusion protein was eluted with an elution buffer (50 mMTris-HCl, 20 mM glutathione, pH 8.0). The eluted fraction was subjectedto exchange with the standard buffer by ultrafiltration. The solutionsthus obtained were treated as final purified samples.

[Example 10] Analysis of Affinity of Single-Domain Variants toMonoclonal IgG-Fab

The single-domain variants obtained in Example 9 were measured foraffinity for the monoclonal IgG-Fab by Biacore in the same manner as inExample 7 to analyze the changes of the binding ability to IgG-Fabcaused by the introduced mutations. Here, only sensorgrams of thesingle-domain variants at a protein concentration of 4 μM (at which thevariants had the same absorbance at 280 nm) were determined.

FIG. 2 show the resulting IgG-Fab binding sensorgrams. The proteinsshowed significantly reduced binding responses to the monoclonal IgG-Fabcompared with that of C-G29A.1d (Comparative Example 3) and theirresponses were reduced to undetectable levels. Thus, the mutationsobtained in the present invention were found to reduce the Fab bindingability. Here, since the responses were reduced to undetectable levels,their affinity constants were not calculated.

[Example 11] Evaluation of Alkali Resistance of C-G29A/S33E.5d

C-G29A/S33E.5d obtained in Example 4 was evaluated for alkali resistanceby comparing decreases in the binding amount to the human IgG (remainingbinding activity to the human IgG) before and after incubation underalkaline conditions for a predetermined period.

Specifically, the binding amount of C-G29A/S33E.5d to the human IgG wasmeasured using the Biacore 3000 before and after an alkali treatment. Inthe alkali treatment, to a 26.2 μM sample of the protein (10 μL) wasadded a certain amount of 0.625 M NaOH to a final concentration of 0.5M. The mixture was incubated for 8 hours at 30° C. Subsequently, 0.5 MHCl (in a certain amount that had been confirmed to neutralize the pH)was added to the treated solution to neutralize the solution. Thesolution was then diluted to ½ in a running buffer (20 mMNaH₂PO₄—Na₂HPO₄, 150 mM NaCl, 0.005% P-20, pH 7.4). In this manner, aC-G29A/S33E.5d solution after the alkali treatment was prepared.

In order to achieve the same protein concentration and the same solutioncomposition as those of the above solution, a C-G29A/S33E.5d solutionbefore the alkali treatment was prepared by preparing a mixed solutionof the NaOH solution used for the alkali treatment and the HCl solutionused for the neutralization treatment in advance, and adding the mixedsolution to a 26.2 μM sample of C-G29A/S33E.5d (10 μL). Preparation of asensor chip (e.g. immobilization of the human IgG), the running bufferused for the measurement, the measurement temperature, and theregeneration treatment of the chip were the same as those in Example 7.Each of the C-G29A/S33E.5d solutions before and after the alkalitreatment was added on the sensor chip at a flow rate of 20 μL/min for150 seconds. A binding reaction sensorgram was then sequentially plottedduring the addition (binding phase, 150 seconds) and after the addition(dissociation phase, 210 seconds).

The analysis was conducted in the same manner as in Example 5. Here, anadditional interpretation of the obtained binding parameters isprovided. In this analysis, the protein concentrations of the solutionsbefore and after the alkali treatment were the same, but theconcentrations of the protein having binding activity to the human IgGwere different from each other. However, since fitting using theconcentration as a variable is difficult, each concentration wasconsidered to be the same before and after the treatment in the fittinganalysis. In this case, the concentration difference of the proteinhaving binding activity to IgG is reflected on the parameter R_(max),which is the maximum binding capacity. Therefore, the alkali resistanceof C-G29A/S33E.5d was evaluated by calculating and comparing therelative value of the R_(max) after the alkali treatment to the R_(max)before the alkali treatment (remaining IgG binding activity (%)).

C-G29A.5d (Comparative Example 1) had a remaining IgG binding activityafter the alkali treatment of 85.4%, whereas C-G29A/S33E.5d had aremaining IgG binding activity after the alkali treatment of 85.5%. Theresults demonstrate that the mutation in the present invention canproduce the effect without sacrificing excellent alkali resistance ofC-G29A.

[Example 12] Preparation of Affinity Separation Matrices with ObtainedProteins (Ligands) Immobilized Thereon Affinity Separation Matrix (1):Methacrylate Polymer-Based

As a water-insoluble base material, a commercial activated filler foraffinity chromatography “TOYOPEARL AF-Formyl-650M” (Tosoh Corporation)was used. This filler was a methacrylate polymer-based filler alreadybearing formyl groups for immobilization of a proteinic ligand. Thefinal purified sample of C-G29A/S33E.5d obtained in Example 4 was usedas a ligand and immobilized to prepare affinity separation matrix (1).

More specifically, 5 mL of the filler was subjected to exchange with acitric acid buffer (0.25 M trisodium citrate dihydrate, pH 9.0 adjustedwith NaOH) on a glass filter, and the volume was increased to 7.5 mL intotal in a centrifuge tube. To this was added 0.64 mL of theC-G29A/S33E.5d-containing solution (64.6 mg/mL), and the resultingmixture was shaken with a mix rotor (MIX ROTOR MR-3 1-336-05 availablefrom AZONE) at 6° C. for 4 hours. Subsequently, the mixture was adjustedto pH 3 with a 2.4 M citric acid aqueous solution and continuouslyshaken at 6° C. for 4 hours. Then, 2.8 mL of a 5.5% by weightdimethylamine borane aqueous solution (Wako Pure Chemical Industries,Ltd.) was added thereto, and the resulting mixture was shaken at 25° C.for 18 hours. The thus prepared carrier was washed on a glass filterwith RO water until the electric conductivity of the washing filtratefell to 5 pS/cm or less. In this manner, the affinity separation matrixwas prepared.

Affinity Separation Matrix (2): Cross-Linked Agarose-Based

As a water-insoluble base material, a commercial 1-mL prepackedactivated column “Hitrap NHS activated HP” (GE Healthcare, Japan) wasused. This column was a cross-linked agarose-based column alreadybearing N-hydroxysuccinimide (NHS) groups for immobilization of aproteinic ligand. The final purified sample of C-G29A/S33E.4d obtainedin Example 4 was used as a ligand and immobilized in accordancegenerally with the product manual to prepare affinity separation matrix(2).

More specifically, the final purified sample was diluted to a finalconcentration of about 6 mg/mL in a coupling buffer (0.2 M sodiumcarbonate, 0.5 M NaCl, pH 8.3) to prepare a sample diluted solution (1mL). The procedure of allowing 2 mL of 1 mM HCl cooled in an ice bath toflow through the column at a flow rate of 1 mL/min was carried out threetimes to remove isopropanol in the column. Then, 1 mL of the samplediluted solution prepared above was immediately added at the same flowrate. The top and bottom of the column were sealed, and the column wasthen left at rest at 25° C. for 30 minutes. In this manner, the obtainedprotein was immobilized on the column. Thereafter, the column wasopened, and 3 mL of the coupling buffer was allowed to flow therethroughat the same flow rate to recover unreacted protein. Next, the procedureof allowing 2 mL of a blocking buffer (0.5 M ethanolamine, 0.5 M NaCl,pH 8.3) to flow through the column was carried out three times, and theprocedure of allowing 2 mL of a washing buffer (0.1 M acetic acid, 0.5 MNaCl, pH 4.0) to flow therethrough was also carried out three times.Finally, 2 mL of a standard buffer (20 mM NaH₂PO₄—Na₂HPO₄, 150 mM NaCl,pH 7.4) was allowed to flow therethrough. Thus, the preparation of theaffinity separation column was completed.

Affinity Separation Matrix (3): Cellulose-Based

As a water-insoluble base material, a commercial gel-filtration filler“Cellulofine GCL-2000-m” (Chisso Corporation) was used. This filler wasa cross-linked porous cellulose-based filler. The final purified sampleof C-G29A/S33E.5d obtained in Example 4 was used as a ligand andimmobilized to prepare affinity separation matrix (3).

More specifically, 12 mL of the filler was subjected to exchange with acitric acid buffer (0.01 M trisodium citrate dihydrate-citric acidmonohydrate, pH 3) on a glass filter (17G-2 available from TOP), and thefluid volume was increased to 18 mL in a centrifuge tube. To this wasadded 6 ml of an aqueous solution containing 0.08 g of sodium periodate(Wako Pure Chemical Industries, Ltd.) dissolved in RO water, and theresulting mixture was shaken with a mix rotor (MIX ROTOR MR-3 1-336-05from AZONE) at 6° C. for about 30 minutes. The thus prepared product waswashed on a glass filter with a sufficient amount of RO water, therebyproviding a carrier bearing formyl groups.

Then, 5 mL of the formyl group-bearing carrier was subjected to exchangewith a citric acid buffer (0.25 M trisodium citrate dihydrate, pH 8.0adjusted with NaOH) on a glass filter, and the volume was increased to8.5 mL in total in a centrifuge tube. To this was added 0.64 mL of theC-G29A/S33E.5d-containing solution (64.6 mg/mL), and the mixture wasadjusted to pH 12 with 0.4 M NaOH and then shaken with the mix rotor at6° C. for 4 hours. Subsequently, the resulting mixture was adjusted topH 3 with a 2.4 M citric acid aqueous solution (citric acid monohydrate)and then continuously shaken at 6° C. for 4 hours. Subsequently, 2.8 mLof a 5.5% by weight dimethylamine borane aqueous solution was addedthereto, and the resulting mixture was shaken at 25° C. for 18 hours.The thus prepared carrier was washed on a glass filter with RO wateruntil the electric conductivity of the washing filtrate fell to 5 pS/cmor less. In this manner, the affinity separation matrix was prepared.

Affinity Separation Matrix (4): Cellulose-Based 2

As a water-insoluble base material, crystalline highly cross-linkedcellulose (gel available from Chisso Corporation, disclosed in U.S. Pat.No. 0,062,118 (JP 2009-242770 A)) was used. The final purified sample ofC-G29A/S33E.5d obtained in Example 4 was used as a ligand andimmobilized to prepare affinity separation matrix (4).

More specifically, 12 mL of the gel was subjected to exchange with acitric acid buffer (0.01 M trisodium citrate dihydrate-citric acidmonohydrate, pH 3) on a glass filter, and the fluid volume was increasedto 18 mL in a centrifuge tube. To this was added 6 ml of an aqueoussolution containing 0.08 g of sodium periodate dissolved in RO water,and the resulting mixture was shaken with the mix rotor at 6° C. forabout 30 minutes.

Then, 5 mL of this formyl group-bearing carrier was subjected toexchange with a citric acid buffer (0.25 M trisodium citrate dihydrate,pH 8.0 adjusted with NaOH) on a glass filter, and the volume wasincreased to 8.5 mL in total in a centrifuge tube. To this was added0.96 mL of the C-G29A/S33E.5d-containing solution (64.6 mg/mL) obtainedin Example 4, and the mixture was adjusted to pH 12 with 0.4 M NaOH andthen shaken with the mix rotor at 6° C. for 4 hours.

Subsequently, the resulting mixture was adjusted to pH 5 with a 2.4 Mcitric acid aqueous solution (citric acid monohydrate) and thencontinuously shaken at 6° C. for 4 hours. Subsequently, 0.46 mL of a5.5% by weight dimethylamine borane aqueous solution was added thereto,and the resulting mixture was shaken at 25° C. for 18 hours. After thereaction, the reaction solution was measured for absorbance at theabsorption maximum around 275 nm. The result revealed that the amount ofC-G29A/S33E.5d introduced was 11 mg/mL-gel, and the yield of the ligandimmobilized on the carrier was 90%.

This carrier was washed on a glass filter with RO water until theelectric conductivity of the washing filtrate fell to 5 μS/cm or less.Then, the carrier was further washed with a 0.1 M citric acid aqueoussolution (citric acid monohydrate), a sodium hydroxide/sodium sulfatemixture aqueous solution (0.05 M NaOH, 0.5 M sodium sulfate), and acitric acid buffer (0.5 M trisodium citrate dihydrate-citric acidmonohydrate, pH 6) in this order. Finally, the carrier was washed withRO water until the electric conductivity of the washing filtrate fell to5 μS/cm or less. In this manner, the affinity separation matrix wasprepared.

This affinity separation matrix (4) was measured in accordance with themethod described in WO 2010/064437 to determine the adsorption capacityfor an antibody and the amount of ligand leakage. The 5% dBC values atcontact times of 1.8 minutes and 3 minutes were 33 and 41 mg/mL,respectively, and the amount of ligand leakage was 30 ppm relative toeluted IgG. Here, the corresponding 5% dBC values of a commercial highlycross-linked agarose carrier “MabSelect” (GE Healthcare Bioscience)measured under the same conditions were 28 and 37 mg/mL, respectively.FIG. 3 shows a graph for comparing the 5% dBC values. The amounts ofligand leakage of commercial affinity separation matrices have beenreported in a known literature (HahnR. et al., “J. Chromatogr. A.”,2006, vol. 1102, pp. 224-231). As described above, the affinityseparation matrix (4) obtained in this example was found to haveperformance parameters important for antibody drug purification atlevels sufficient for practical use.

[Example 13] Evaluation of Antibody Elution Properties of AffinitySeparation Matrices with Acid

An empty column Tricorn™ 5/50 Column (GE Healthcare, Japan) was filledwith each of the affinity separation matrices (1) to (4) prepared inExample 12, and the column was connected to a chromato system AKTA primeplus (GE Healthcare, Japan) to evaluate the antibody elution propertiesin the acidic condition by antibody purification chromatography. Here,the affinity separation matrix (2), which was a prepacked column, couldbe connected as it was. All the columns had a volume of about 1 mL.After equilibration of each affinity separation column with a standardbuffer (20 mM NaH₂PO₄—Na₂HPO₄, 150 mM NaCl, pH 7.4), 500 μL of a 1 mg/mLsolution of herceptin (humanized monoclonal IgG drug of the VH3subfamily) in the standard buffer was added thereto at a flow rate of 2mL/min. Subsequently, the column was washed with 5 mL of the standardbuffer flowing at the same flow rate, and then 5 mL of an elution buffer(35 mM CH₃COOH—CH₃COONa, pH 3.5) was also allowed to flow therethroughat the same flow rate. During this period, the absorbance at 280 nm wasmonitored to determine a chromatographic profile of an IgG elution peak.Continuously, 5 mL of the standard buffer was allowed to flowtherethrough at the same flow rate, and then 3 mL of a strong washingsolution (0.5 M CH₃COOH, 0.1 M Na₂SO₄, pH 2.5) was also allowed to flowat the same flow rate. During this period, the absorbance at 280 nm wasmonitored to determine a peak of forcibly separated IgG which had beenstill present in the column. Here, only for the affinity separationmatrix (2), a strong washing solution (20 mM CH₃COONa—CH₃COOH, 1M NaCl(pH 3.2)) was used.

The affinity separation matrices (1) to (3) were evaluated for antibodyelution properties in the acidic condition by carrying out antibodypurification chromatography with an elution buffer adjusted to pH 3.75in the same manner as described above.

Antibody Elution Properties with Acid of Affinity Separation Matrix (1)

FIG. 4 show graphs on which elution peak profiles obtained by antibodypurification chromatography using the affinity separation matrix (1)with C-G29A/S33E.5d or with C-G29A.5d (Comparative Example 1)immobilized instead are superimposed. The upper graph shows elution peakprofiles obtained at an elution pH of 3.5, and the lower graph showselution peak profiles obtained at an elution pH of 3.75. As shown inFIG. 4, the elution peak profiles obtained using the matrix withC-G29A/S33E.5d immobilized thereon were apparently sharper than thoseobtained using the matrix with C-G29A.5d immobilized thereon. From theelution peak profiles at an elution pH of 3.75, it was demonstrated thatrecovery of IgG adsorbed on C-G29A.5d, which corresponds to a proteinbefore introduction of the mutation, was difficult but IgG adsorbed onC-G29A/S33E.5d, which corresponds to a protein after introduction of themutation, could be all recovered. This may be one example showing thatthe present invention can drastically enhance the antibody recovery ratein the presence of an acidic solution closer to neutral.

Antibody Elution Properties of Affinity Separation Matrix (2) with Acid

FIG. 5 show graphs on which elution peak profiles obtained by antibodypurification chromatography using the affinity separation matrix (2)with C-G29A/S33E.4d or with C-G29A.4d (Comparative Example 2)immobilized instead are superimposed. Likewise, as seen in the case ofthe affinity separation matrix (1), the elution peak profiles obtainedusing the matrix with C-G29A/S33E.4d immobilized thereon were apparentlysharper than those obtained using the matrix with C-G29A.4d immobilizedthereon. The obtained data demonstrated that the protein of the presentinvention can produce the effect regardless of the type of the base ofthe water-insoluble base material, the immobilization method of theligand on the base material, and the number of domains of the proteinserving as a ligand.

Antibody Elution Properties of Affinity Separation Matrix (3) with Acid

FIG. 6 show graphs on which elution peak profiles obtained by antibodypurification chromatography using the affinity separation matrix (3)with C-G29A/S33E.5d or with C-G29A.5d (Comparative Example 1)immobilized instead are superimposed. It was shown that the elution peakprofiles obtained using the matrix with C-G29A/S33E.5d immobilizedthereon were apparently sharper than those obtained using the matrixwith C-G29A.5d immobilized thereon. The obtained data demonstrated thatthe protein of the present invention can produce a better effect when abase material with excellent antibody elution properties (sharp elution)is used in combination.

Antibody Elution Properties of Affinity Separation Matrix (4) with Acid

FIG. 7 shows an elution peak profile (elution pH: 3.5) obtained byantibody purification chromatography using the affinity separationmatrix (4) with C-G29A/S33E.5d immobilized thereon. From an industrialpoint of view, a need for matrices having a high antibody-bindingcapacity is large. Increase in the amount of immobilized ligands iseffective to increase the antibody-binding capacity. The obtained datademonstrated that the present invention can produce the effect withoutcausing any problems even when the amount of immobilized proteins ishigh.

[Comparative Example 1] Preparation and Use of C-G29A.5d

C-G29A.5d (SEQ ID No:28) was prepared using the recombinant cellsobtained in Example 1 in the same manner as in Examples 3 to 5. Theaffinities for the human IgG and the monoclonal IgG-Fab were analyzed inthe same manner as in Examples 5 and 7, and the analysis results arealso shown in Tables 1 and 2.

The alkali resistance was also evaluated in the same manner as inExample 11. Additionally, C-G29A.5d was used instead for preparation ofthe affinity separation matrices (1) and (3) in Example 12, and theresulting matrices were evaluated for antibody elution properties in theacidic condition in the same manner as in Example 13. The results arealso shown in FIGS. 4 and 6. Here, the solutions used for preparation ofthe affinity separation matrices (1) and (3) had a protein concentrationof 58.2 mg/mL, and the used amount was 0.79 mL.

[Comparative Example 2] Preparation and Use of C-G29A.4d

An expression plasmid for C-G29A.4d and transformant cells obtainedusing the plasmid were prepared using the expression plasmidpNK3262-C-G29A.5d obtained in Example 1 as a template and theoligonucleotide primers of SEQ ID Nos:66 and 67 in the same manner as inExample 2. C-G29A.4d was prepared in the same manner as in Examples 3 to5. Additionally, C-G29A.4d was used instead for preparation of theaffinity separation matrix (2) in Example 12, and the resulting matrixwas evaluated for antibody elution properties in the acidic condition inthe same manner as in Example 13. The results are also shown in FIG. 5.

[Comparative Example 3] Preparation of C-G29A.1d

C-G29A.1d was prepared using the transformant cells (HB101) containingthe template plasmid used in Example 8 in the same manner as in Example9, and the affinity for the monoclonal IgG-Fab was analyzed in the samemanner as in Example 10. The analysis results are also shown in FIG. 2.

1-25. (canceled)
 26. A method for separating and purifying an antibody,the method comprising: adsorbing an immunoglobulin comprising a Fabregion onto an affinity separation matrix; and eluting the antibodyadsorbed onto the affinity separation matrix with an acidic solution,wherein the affinity separation matrix comprises a carrier and a ligandimmobilized on the carrier, wherein the ligand is a protein comprisingan amino acid having a sequence identity of not less than 90% to theamino acid sequence of SEQ ID NO:5, and wherein the ligand comprises atleast one substitution selected from the group consisting of: asubstitution for Ser at position 33 of SEQ ID NO:5; a substitution forLys at position 35 of SEQ ID NO:5; a substitution for Asp at position 36of SEQ ID NO:5; and a substitution for Asp at position 37 of SEQ IDNO:5, and wherein the ligand has an affinity for the Fab region lowerthan an affinity for the Fab region that a protein comprising the aminoacid sequence of SEQ ID NO:5 has.
 27. The method according to claim 26,wherein the substitution for Ser at position 33 of SEQ ID NO: 5 is asubstitution of Glu, Leu, or Thr, wherein the substitution for Lys atposition 35 of SEQ ID NO: 5 is a substitution of Arg, wherein thesubstitution for Asp at position 36 of SEQ ID NO: 5 is a substitution ofArg or Ile, and wherein the substitution for Asp at position 37 of SEQID NO: 5 is a substitution of Glu.
 28. The method according to claim 26,wherein the pH of the acidic solution is 3.0-5.0.
 29. The methodaccording to claim 26, wherein the carrier comprises a water-insolublebase material.
 30. The method according to claim 29, wherein thewater-insoluble base material comprises a synthetic polymer or apolysaccharide.
 31. The method according to claim 30, wherein thewater-insoluble base material comprises the polysaccharide, and thepolysaccharide is cellulose.
 32. The method according to claim 31,wherein the immunoglobulin is immunoglobulin G or an immunoglobulin Gderivative, and wherein the immunoglobulin G derivative is selected fromthe group consisting of: chimeric immunoglobulin G; humanizedimmunoglobulin G; and immunoglobulin G comprising an Fc region with amodified sugar chain.
 33. The method according to claim 26, wherein theligand further comprises a substitution of Ala for Gly at position 29 ofSEQ ID NO:5.
 34. The method according to claim 33, wherein the ligandcomprises the substitution of Glu, Leu, or Thr for Ser at position 33 ofSEQ ID NO:5.
 35. The method according to claim 33, wherein the ligandcomprises the substitution of Arg or Ile for Asp at position 36 of SEQID NO:5.
 36. The method according to claim 33, wherein the ligandcomprises the substitution of Arg for Lys at position 35 of SEQ ID NO:5and the substitution of Glu for Asp at position 37 of SEQ ID NO:5.